The central goal of our work is to use embryonic stem (ES) cells as a source from which to derive therapeutically relevant skeletal progenitor cells to treat conditions associated with bone and cartilage damage. The field of cell based therapeutics is largely at its infancy and enormous challenges lie ahead, including the ability to isolate specific cell types, maintain cells appropriately in culture, differentiate cells down a restricted lineage, and assess engrafted cell function after transplantation. There is an absolute need within this field to generate the necessary research tools to aid investigators in resolving these challenges. The process of differentiating ES cells into cartilage and bone, like embryonic development, will involve several intermediate differentiation steps and currently it is unknown at what point in the differentiation process will the ideal progenitor cell population(s) exist. Therefore, it will be critical to test and compare different progenitor cell populations that transiently form within a lineage for their capacity to differentiate into chondrocytes and osteoblasts upon transplantation. The aims of this proposal are focused on identifying and testing progenitor cell types of the axial skeleton lineage. To carry this out, we propose to generate fluorescent protein reporter ES cell lines that will mark progenitor and mature skeletal cell types. Aim1 focuses on generating a three fluorescent protein reporter ES cell line to distinctly identify cell types for early mesoderm, presomitic paraxial mesoderm, and sclerotome allowing us to study axial skeletal progenitor cell formation. Aim2 will generate ES cell lines that will couple different axial skeletal progenitor markers with a chondrocyte/osteoblast ES cell line thus enabling us to test and compare the ability to differentiate progenitor cell types to differentiate into cartilage and bone cells upon transplantation. PUBLIC HEALTH RELEVANCE: The aims of this application are focused on the study and testing of embryonic stem (ES) cell derived axial skeletal progenitor cells. We propose to generate novel ES cell lines that use fluorescent protein reporters to aid in the identification of progenitor and mature skeletal cell types of the cartilage and bone lineage. These ES cell lines will allow us to study the formation of axial skeletal progenitor cells and test their ability to differentiate into bone and cartilage upon transplantation. The reagents developed from this grant will have an important impact on therapeutic approaches involving the use ES cells to repair damaged cartilage and bone.